Somatic cell derived embryonic stem cells and its differentiated cells

ABSTRACT

The present invention discloses a process for obtaining somatic cell derived embryonic stem cells (encoded by reprogrammed somatic cell nuclei), ES cell-like cells or other types of embryo-derived stem cells by nuclear transplantation, and a process for inducing said stem cells into various differentiated cell types.

TECHNICAL FIELD

The present Invention generally relates to the preparation of somaticcell derived embryonic stem cells (S-ES cells, also termed as nucleartransfer embryonic stem cells, ntES cells), or embryonic stem-like cellsor other types of embryo-derived stem cells from nuclear transfer (nt)units (nt-units) at various implantation stages, by transplantation ofhuman somatic cells or their nuclei into enucleated animal oocytes, andin a preferred embodiment the rabbit enucleated oocytes. The presentinvention more specifically relates to the preparation human ntES cells,embryonic stem-like cells or other types of embryo-derived stem cells bytransplanting the human cells or cell nuclei into enucleated animaloocytes, more preferable leporid oocytes and most preferable enucleatedNew Zealand rabbit oocytes.

The present invention further relates to the use of ntES cells,embryonic stem-like cells or other types of embryo-derived stem cells inthe induction of differentiated cells. ntES cells, embryonic stem-likecells, or other types of embryo-derived stem cells, eitherdifferentiated or non-differentiated, can be modified and used as cellcarriers to introduce various types of bio-active molecules, includingDNAs, RNAs or protein etc., into the human body. ntES cells, embryonicstem-like cells, or other types of embryo-derived stem cells, bothmodified and unmodified, can be used in the production of all kinds ofdifferentiated cells, tissues and organs for the treatment and diagnosisof diseases. Also, the cells, of which the genes have been changed orunchanged, can per se be used as nuclear donors in the nucleartransplantation.

BACKGROUND OF THE INVENTION

The nuclear transplantation involves the transplantation of donor cellsor cell nuclei into enucleated oocytes. The resultant nt-units candevelop to various pre-implantation stages, or further into life-borneanimals. This method was shown to be successful when applied toamphibians at the end of the 1950s. Briggs and King obtained nucleartransferred frogs by transferring nuclei of the enteric epithelium ofthe rananigromaculata into oocytes. Nuclear transfer had not beenapplied to mammals until the end of 1980s. Many types of somatic cellswere used in the nuclear transfer experiments as the nuclear donorcells, including embryo blastomeres, inner cell mass, and terminalembryo cells in nuclear transfer (Collas et al., Mol. Reprod. Dev.,38:264-267, 1994; Keefer et al., Biology of Reproduction, 50:935-939,1994: Sims et al., PNAS, 90:6155-6159, 1993).

Using adult sheep mammary gland as donor cell, Wilmut et al. in Britain(Nature 1997, 385, 810-813) produced the first living lamb from somaticcell nuclear transfer. In 1998, sequential mice somatic cells nucleartransplantation into was successfully completed in US (Wakayama, et al.Nature 394: 369-374, 1998). In 1999, nuclear transplantation of miceembryonic stem cell (ES) was completed (Teruhiko et al., PNAS96:14984-14989, 1999). The success of nuclear transplantation usingadult somatic cells is not only a progress in technique but also aprogress in concept, showing the possibility that highly differentiatedadult somatic cell nuclei can form new individuals once beingreprogrammed to reenter development

In 1999, Dominko et al. injected somatic cell nuclei from variousanimals (e.g. cows, sheep, pigs, monkeys, and rats) into bovine oocytesto develop nt-units, each developing to some extent (Biology ofReproduction. 60 (6): 1496-1502, 1999). These experiments show thatmammalian somatic nuclei can be activated by oocytes of a speciesdifferent from the nuclear donor to form nt-units. Such nt-units candevelop to all the pre-implantation stages. The finding, that oocytes ofone species can reprogram somatic nuclei of another species, shows thatmechanisms controlling reprogramming are highly conserved in differentmammalian species.

The advancement in ES cell cultivation has been also highlighted allover the world these years while the development of the nuclear transfertechnology is flourishing. The basic manipulation involved in theestablishment of ES cell and the basic characters and the applicationthereof have been well known in the art since the establishment of themice ES cell line in 1981 (See Evans, et al. Nature, 29: 154-156, 1981;Martin, et al. PNAS, 78: 7634-7638, 1981). The ES cell can be kept in anundifferentiated, infinitely proliferating state. providing that thecultivation thereof is effected in a feeder layer of fibroblast cells(Evans, et al.) or under differentiation inhibiting conditions (Smith,et al. Development Biology, 121:1-9, 1987).

ES cells have the potential of development into all cell types of abody, including germ cell. ES cells can be differentiated to variousspecific cell types under appropriate induction conditions. Embryonicstem cells have been successfully directed to differentiate in vitrointo various types of cells, e.g. the hematopoletic stem cells (Ronald,et al. PNAS, 92: 7530-7534, 1995), neural cells (Dinsmore, et al.Theriogenology, 49: 145-151, 1998), muscle cells (Reubinoff, et al.Nature Biotechnology, 18 (4): 399-404, 2000), adipocytes (Dani C Smith,et al. J Cell Sci, 110: 1279-1285, 1997), endothelial cells (Vittet, etal. Blood, 88 (9): 3424-3431, 1996) and so on. A specific cell type,e.g. a muscle-like cell, differentiated from ES cells display propertiessimilar to that of its natural equivalent cell types, e.g. a musclecell, therefore, cells differentiated from ES cell can be use intreatment of diseases (cell, tissue, or organ transplantation).

In view of the potentiality of the mouse ES cells, it has been tried toculture the ES cells of large mammals because the establishment thereofnot only has the significance in scientific research but also can beapplied to medicine. For example, the human ES cells can be directed toall kinds of specialized cells for the treatment of diseases. Because oftheir proliferation and differentiation potential, ES cells provided aplatform for genetic modification. ES cells of large animals can begenetically modified to produce various biological products.

Isolation of ES cells or embryonic stem-like cells from large mammalshave been reported. For example, Notarianni, et al. (J. Reprod. Fert.,Suppl. 43: 255-260, 1991) reported that the cells in primary cultures ofinner cell masses from pig and sheep blastocysts exhibit somemorphological and growth characteristics similar to ES cells. Chen R L,et al. (Biology of Reproduction, 57 (4): 756-764, 1997) and Wianny, etal. (Theriogenology, 52 (2): 195-212, 1999) reported the isolation ofpig ES cells from porcine blastocysts, respectively,

Stekelenburg-Hamers, et al. reported the isolation and thecharacterization of embryonic stem-like cells from inner cell mass ofbovine blastocysts (Mol. Reprod. 40: 444-454, 1995).

Thomson, et al. reported the successful isolation of ES cells fromprimate macaque (PNAS, 92 (17): 7844-8, 1995).

Thomson, et al. successfully established human ES cells lines (Science,282 (6): 1145-1147, 1998), which is an important breakthrough in thestem cells research. These call lines not only can be used as importanttools in the research of human development, but also has the broadapplication prospect in medical fields. For example, (1) human ES cellslines can be expanded and differentiated into specific cell types tomeet the needs of the patients. They will become the cell source forcell or organ transplantation therapies. It is possible that many humandiseases can be treated through cell transplantation. Besides itsmedical applications, (2) human ES cell lines may also facilitate thescreen for new drugs and the safety evaluation of drugs.

However, the cells differentiated from the human ES cells may causeimmune rejection while being used in the transplantation between theindividuals of different MHC types, thus the patient would have to takeimmune inhibitor, which is toxic. At present, there is no method inobtaining ES cells that are compatible with the patient's immune systemsby using his somatic cells.

Munsie, at al. reported the isolation of mice ES cells from blastocystsderived by somatic cell nuclear transfer (Current Biology 10: 989-992,2000). Wakayama, et al. obtained the mice ES cells, which can be inducedto various types of specific cells in vitro, from the cultures ofblastocysts derived by somatic cell nuclear transfer (Science, 292(5517); 740-743. 2001). The result of the research done by Wakayama, etal. demonstrates that ES cells can be isolated from nuclear transferembryos by somatic cell nuclear transfer. The ntES cells of somatic cellorigin are pluripotent and can differentiate into any specific celltypes as ES cells derived from the normal zygote.

The successes achieved by all of these scientists mentioned aboveestablished a new route for treatment of the diseases, i.e., therapeuticcloning. It is suggested that somatic cells of the patient can bereprogrammed through nuclear transfer to produce ntES cells. The ntEScells obtained are further differentiated into the specific cell typeneeded by the patient. ntES cells and their differentiated progenieshave the same genotype as the patient, and therefore would not berejected by the patient's immune system when transplanted to thepatient. Therapeutic cloning provides an approach to solve the problemof immune rejection commonly observed in transplantation medicine.

WO 98/07841 (Robe, et al., Massachusetts, U.S.A., filed in 1998)disclosed the isolation of the thirty 2-cell-stage embryos and six 4- to16-cell-stage embryos and one 16- to 400-cell-stage embryo from theallogeneic nuclear transplantation from lymphocyte and mouth epitheliumof human to the bovine oocytes. However, this study failed to provideany proof demonstrating that the embryos and cell colonies derived fromthe embryos were encoded by human genomic DNA rather than bovine genomicDNA. Blastocysts can be easily created through parthenogenesis of bovineoocytes. Furthermore, the patent application provided no proofdemonstrating that the colonies were encoded by human genomic DNA anddisplayed any characteristics of human or primate stem cells,

Up to the submission of the present application, there has been noreport that the ntES cells can be obtained by human somatic cell nucleitransfer, neither the report that human specific cell types can bedifferentiated therefrom.

CONTENTS OF THE INVENTION

The present inventors discovered that nuclear transfer (nt) units(nt-units) could be obtained by transplantation of human cells, such asadult somatic cells or cell nuclei, into enucleated mammalian (includinghuman) oocytes. The nuclear transfer embryonic stem cells (ntES cells)could be obtained from the nt-units at various pre-implantation stages.The ntES cells are similar in cellular characters and differentiationpotential to that of the human ES cells obtained from the fertilizedzygotes. The result was the first proof that human somatic cell nucleimight be reprogrammed after the transplantation into the enucleatedoocytes. The result also proved that the human embryonic stem cellscould be derived from nt-units at the blastocyst stage.

The result further proved the feasibility of cross-species nucleartransplantation, e. g. the transplantation of human cells or cell nucleiinto the enucleated oocytes of a leporid animal, e.g. rabbit, to producent-units, which when cultured under appropriate conditions give rise tont-units at various developmental stages encoded by the donor nuclei.

Therefore, it Is an object of the invention to provide a method ofobtaining ntES cells, embryonic stem-like cells or other types ofembryo-derived stem cells. The method comprises reprogramming humansomatic cell nuclei to obtain nt-units through nuclear transfer, andisolating ntES cells, embryonic stem-like cells or other types ofembryo-derived stem cells from nt-units at various pre-implantationstages.

It is another object of the invention to provide improved methods forcross-species somatic cell nuclear transplantation.

It is a specific object of the invention to provide a novel method forproducing ntES cells, embryonic stem-like cells or other types ofembryo-derived stem cells, involving the transplantation of the cells orcell nuclei of a mammalian species (including human) into enucleatedoocytes of a species different from the nuclear donor.

It is another object of the invention to provide a novel method forproducing human ntES cells, embryonic stem-like cells or other types ofembryo-derived stem cells, involving the transplantation of the cells,e.g. human adult somatic cells or cell nuclei into enucleated oocytes ofthe same species, e.g. human oocytes.

It is another object of the invention to produce human ntES cells,embryonic stem-like cells or other types of embryo-derived stem cells bytransplantation of human cells or cell nuclei into enucleated oocytes,e.g. enucleated leporid oocytes.

It is another object of the invention to provide a novel method forproducing ntES cells, embryonic stem-like cells or other types ofembryo-derived stem cells, involving the transplantation of the cells orcell nuclei of a human into enucleated oocytes, e.g. enucleated rabbitoocyte.

It is a more specific object of the invention to obtain ntES cell linesthat could proliferate without limiting in a similar way to theembryonic stem cells obtained from fertilized embryos, which shouldexpress all the special markers of the primate ES cells, and have thepotential to differentiate to all kinds of cells of ectoderm, mesoderm,and endoderm.

It is a specific object of the Invention to obtain the ntES cell lines,which are compatible to the immune system of the nuclear donor. The ntEScells and the cells, tissues and organs derived from ntES cells areencoded by the genome of the nuclear donor, Thus, transplantation of thentES cells or cells, tissues and organs derived from ntES cells backinto the nuclear donor, e.g. the patient, will not cause immunerejection.

It is a more specific object of the invention to differentiate humanntES cells, embryonic stem-like cells or other types of embryo-derivedstem cells directionally to specific type cells including muscle cells,neural cells, fibroblasts and adipocytes in a specific inductiveenvironment, including in vivo and in vitro inductive system.

It is another specific object of the invention to use the human ntEScells or embryonic stem-like cells or other types of embryo-derived stemcells and their differentiated progenies for the treatment and diagnosisof diseases.

It is another specific object of the invention to use human ntES cells,embryonic stem-like cells or other types of embryo-derived stem cellsand their differentiated progenies for construction of differentiatedhuman tissues or organs.

It is another object of the invention to provide human ntES cells, orembryonic stem-like cells, or other types of embryo-derived stem cellsor differentiated cells, tissues or organs derived through somatic cellnuclear transfer for transplantation therapies. Such therapies includeby way of examples the treatment of diseases and injuries, including butnot limited to Parkinson's syndrome, Huntington's syndrome, Alzheimer'ssyndrome, ALS, spinal cord injuries, multiple sclerosis, musculardystrophy, diabetes, liver disease, heart disease, cartilagereplacement, burns, vascular disease, urinary tract disease, as well asthe treatment of immunodeficiency diseases, bone marrow transplantation,cancer, etc.

It is another specific object of the invention to use the human ntEScells, embryonic stem-like cells or other types of embryo-derived stemcells and their differentiated progenies as cell carriers for thetransport of all kinds of bio-active molecule, modified DNA, RNA orprotein etc. into the human body. The cells, modified or unmodified, maybe used for the preparation of all kinds of differentiated cells,tissues and organs for use in medical therapies, including the treatmentand diagnostic of diseases.

It is another object of the invention to use human ntES cells, embryonicstem-like cells or other types of embryo-derived stem cells and theirdifferentiated progenies in the treatment of various diseases, inparticular for the treatment and/or prevention of the diseases andInjuries specified, supra.

It is another object of the invention to use genetically modified humanntES cells, embryonic stem-like cells or other types of embryo-derivedstem cells and their differentiated progenies as nuclear donors in thenuclear transplantation.

It is another specific object of the invention to use human ntES cells,embryonic stem-like cells or other types of embryo-derived stem cellsand their differentiated progenies for the study of cell differentiationand for drug screening and toxicity evaluation.

With the foregoing and other objectives, advantages and features of theinvention that will become hereinafter apparent, the nature of theinvention may be more clearly understood by reference to the followingdetailed description of the preferred embodiments of the invention andto the appended claims.

BRIEFS DESCRIPTION OF THE FIGURES

FIG. 1 is a photograph of the fibroblasts obtained from a 42-year-oldman foreskin.

FIG. 2 is a photograph of the rabbit oocytes.

FIG. 3 is a photograph of the nt-unit (obtained by injection of asomatic cell into the zona pellucid) at the 4cell stage.

FIG. 4 is a photograph of the nt-unit (obtained by injection of asomatic cell into the zona pellucid) at the morular stage.

FIG. 5 is a photograph of the nt-unit (obtained by injection of asomatic cell into the zone pellucid) at the blastocyst stage.

FIG. 6 is a photograph of the nt-unit (obtained by injection of asomatic cell into the zona pellucid) at the hatching blastocyst stage.

FIG. 7 is a photograph of the nt-unit (obtained by injection of thesomatic cell into the cytoplasm of the oocyte) at the 4-cell stage.

FIG. 8 is a photograph of the nt-unit (obtained by injection of thesomatic cell into the cytoplasm of the oocyte) at the morula stage.

FIG. 9 is a photograph of the nt-unit (obtained by injection of thesomatic cell into the cytoplasm of the oocyte) at the blastocyst stage.

FIG. 10 is a photograph of the nt-unit (obtained by injection of thesomatic cell into the cytoplasm of the oocyte) at the hatchingblastocyst stage.

FIG. 11 is a table showing somatic cells from donors at different agesformed blastocysts with comparable efficiency.

FIG. 12 is a photograph of a ntES cell colony derived from human somaticcells reprogrammed by a rabbit oocyte.

FIG. 13 is a photograph of a ntES cell colony derived by human somaticcell nuclear transfer, express markers typical of primate ES calls, andhigh alkaline phosphatase activity.

FIG. 14 shows that ntES cells are capable of forming an embryold body bysomatic cell nuclear transfer.

FIG. 15 is a photograph of muscle cells differentiated from human ntEScells by somatic cell nuclear transfer.

FIG. 16 is a photograph of neurocyte differentiated from human ntEScells by somatic cell nuclear transfer.

FIG. 17 is a photograph of fibroblast like cells differentiated fromhuman ntES cells by somatic cell nuclear transfer.

FIG. 18 is a photograph of adipocytes differentiated from human ntEScells by somatic cell nuclear transfer.

FIG. 19 is a photograph showing that ntES cells are capable ofdifferentiating into cells expressing markers of all three germ layers.

FIG. 20 is a photograph of the karyotype of the ntES cell of the 26^(th)passage.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, nuclear transfer (nt) and nucleartransplantation are used interchangeably.

In the present invention, somatic embryo and nt-unit are usedinterchangeably.

In the present invention, nuclear transfer embryonic stem cell (ntEScell) and somatic cell derived embryonic stem cell (S-ES) are usedinterchangeably.

The term “nuclear transplantation” referred herein means thetransplantation of donor cells or cell nuclei into enucleated oocytes.The resultant nt-units are cultured to various pre-implantation stages(e.g. blastocyst) or are allowed to further develop into live-bonenon-human animals. For nuclear transfer, cells or cell nuclei of humanor animals, for example, animals of primates, ungulates, amphibians,rodents species, can all be used as nuclear donors. Human oocytes may beused in nuclear transplantation, as well as oocytes from other species,including those derived from primates, ungulates, amphibians, rodents,etc.

The term “homogeneous nuclear transplantation” referred herein means thetransplantation of donor cells or cell nuclei into enucleated oocytesfrom the same species. The resultant nt-units are cultured to variouspre-implantation stages or allowed to further develop into live-borneanimals.

The term “nuclear transplantation in different species” referred hereinmeans the transplantation of donor cells or cell nuclei into enucleatedoocytes of a species different from the nuclear donor. The resultantnt-units are cultured to various pre-implantation stages or allowed tofurther develop into live-borne animals.

The term “nuclear transfer (nt) unit (nt-unit)” referred herein means aunit derived from the combination of a nuclear donor and an enucleatedoocyte. The nuclear donor and the enucleated oocyte may be obtained fromthe same species or from different species.

The term “somatic embryo” reffered herein means nt-units at variouspre-implantation stages, including the 2-cell stage, 4-cell stage,8-cell stage, morula stage, blastocyst stage, and hatching blastocyststage.

The term “somatic cell” referred herein means all cells types in anadult body except the germ cells.

For mammalian species, a zygote develops through the 2-cell stage,4-cell stage, 8-cell stage, morula stage, blastocyst, hatchingblastocyst in sequence. Inner cell mass located in a blastocyst is thefounder of the embryo proper, which will give rise to all cells of theembryo. ES cells are the equivalent of cells of the inner cell mass,therefore are pluripotent, capable of development to any of the cells ofthe growing fetus including the germ line.

Human ES cell derived from the inner cell mass is a type of pluripotentstem cells which can be induced to differentiate into any cell types,including germ line cells, and is permanent in vitro. In a long-termculture, these cells maintain a normal karyotype. The cells express thespecial markers: negative for SSEA-1 and positive for SSEA-3, SSEA-4,TRA-1-60, TRA-1-81. They are positive for alkaline phosphatase.

Human nuclear transfer embryonic stem cells, obtained by transplantationof human cell nuclei into non-human mammalian oocytes, have propertiessimilar to ES cells isolated from the fertilized zygote. Human ntEScells can be propagated infinitely in vitro and maintain a normalkaryotype. These cells can be induced to differentiate into cells of allthree germ layers. Undifferentiated human ntES cells express markerstypical of primate ES cells, which are negative for SSEA-1 and positivefor SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and are positive for alkalinephosphatase.

The term “embryonic stem-like cells” referred herein means the cell-massderived from nt-units at greater than the 2-cell stages that has part ofthe characters of the human embryonic stem cells as above.

The term “other types embryo-derived stem cells” referred herein meansall cells derived from nt-units at various pre-implantation stagesexcept embryonic stem cells and embryonic stem-like cells. The term“specialized cells” referred herein means the cells differentiated fromntES cells, embryonic stem-like cells and other types of embryo-derivedstem cells, which can be induced artificially to differentiate in vivoor in vitro.

The present invention provides a method of preparing the ntES cells,embryonic stem-like cells or other types of embryo-derived stem, cells,comprising the following steps:

(i) transplanting nuclear donor cells or cell nuclei into enucleatedoocytes to form nt-units;

(ii) activating the obtained nuclear transfer units;

(iii) culturing the nuclear transfer units to various pre-implantationstages; and

(iv) obtaining nuclear transfer embryonic stem cells, embryonicstem-like cells or other types of embryo-derived stem cells coded by thedonor nuclei from the nt-units greater than 2-cell development stage.

The present invention provides a further method of inducing the ntEScells into many types of specific cells, comprising the following steps:

(i) preparing embryonic stem cells or embryonic stem-like cells or othertypes of embryo-derived stem cells from reprogrammed somatic cells bynuclear transfer; and

(ii) inducing the above cells to differentiate to specialized cellsunder appropriate condition.

In a preferred embodiment, an appropriate small animal, such as arabbit, is used as an oocyte donor for nuclear transfer. By maintainingthese animals in appropriate animal houses, they could be fed withstandard food and would be more easily administered, thus resulting in alower cost. Moreover, it would be easier to control these rabbits freefrom diseases and being SPF (specific pathogen free). Four days afterinjection of a rabbit with artificial hormone, about 30 oocytes can beobtained for the nuclear transfer experiment. Rabbit nature estrusperiod is 7-9 days.

The present inventors discovered that a nuclear transfer unit could beobtained by transplantation of the nuclei of human cells, specificallyhuman fibroblasts, into enucleated rabbit oocytes to obtain nt-units atvarious developmental stages.

In view of the fact that human cell nuclei can be effectivelyreprogrammed by rabbit oocytes, it is reasonable to expect that humansomatic cells may be transplanted into oocytes of other non-rabbitanimal species (e.g. ungulates) to obtain nu-units. Oocytes from otheranimal sources should also be suitable. For example, oocytes derivedfrom non-human primates, amphibians, rodents, etc. Further, usingsimilar methods, it should be possible to transfer human cells or nucleiinto human oocytes and use the resultant blastocysts to produce ntEScells.

Therefore, in its broadest sense, the present invention involves thetransplantation of human or animal cells or cell nuclei into enucleatedoocytes of a species different from the nuclear donor to obtain nt-unitsfor isolating embryonic stem cells or embryonic stem-like cells or othertypes of embryo-derived stem cells. For example, the invention mayinvolve the transplantation of human cells or cell nuclei intoenucleated oocytes of another species (e.g. a rodent) to producent-units, which can develop to nt-units at various pre-implantationstages, including those at the morula stage, blastocytes, and hatchingblastocytes stages. nt-units at various preimplantation stages areuseful for isolation of embryonic stem cells, embryonic stem-like cellsor other types of embryo-derived stem cells for therapeutic cloning.

The present inventors discovered that nuclear transfer units can beobtained by transplantation of human somatic cell nuclei into enucleatedanimal oocytes to produce nt-units at the blastocyst stage. ntES cellscan then be isolated from nt-units at the blastocyst stage. Theseresults demonstrate that the blastocysts obtained from cross-speciesnuclear transplantation have the capability to give rise the embryonicstem cell lines as the embryos derived from fertilized zygotes.

ntES cell lines from cross-species nuclear transfer can be cultured onfeeder cell layers for a long period of time, and can passage for morethan 30 passages. Cells obtained thereby have not only the capability ofdifferentiation into all three germ layers, including ectoderm,mesoderm, endoderm, but also the capability of differentiating to manykinds of specialized cells including muscle cells, adipocytes, nervecell and fibroblast etc.

These discoveries are of great importance in solving the problem ofimmune rejection in medical transplantation. As well known in the art,new organs, cells or tissues can be used to replace or reverse thefunctions of the original organs, cells or tissues when they could notfunction properly. For example, the kidney transplantation can beeffected if a kidney cannot do function normally, and hematopoletic stemcells from other human are used to replace the exhausted hematopoieticstem cells in the therapy of the tumor. It has been a serious problem inthe medical transplantation for several decades that the organs, cellsor tissues transplanted into the patients would be rejected by theimmune system of the recipient, because of the mismatch in MHC genesbetween the organ donor and recipient. In recent years, scientists putforward a conception of therapeutic cloning targeting for solving theimmune rejection. The somatic cells could be reprogrammed by nucleartransplantation in order to obtain nt-units for isolating ntES cells.ntES cells can then be induced to differentiate into the, organs,tissues or cells needed by the patient, and transplanted back into thepatient. Since the resultant cells, tissues or organs are encoded by thepatient's own genome and will most likely be recognized as “self”,transplantation of cells, tissues and organs resulting from therapeuticcloning should not cause immune rejection. The present inventorsdiscovered the following in the invention:

(i) ntES cell lines can be obtained from human somatic cell nuclei bysomatic cell nuclear transfer.

(ii) ntES cell lines can proliferate in vitro for long time asconventional human ES cells derived from fertilized zygotes.

(iii) nt cells have the potential to differentiate into cell types ofall 3 germ layers, including ectoderm, mesoderm, endoderm. Thesediscoveries demonstrate that therapeutic cloning is practicallyfeasible. The present inventors further discovered that human somaticcell could be reprogrammed effectively by the oocyte of a non-humanmammalian species, preferably the oocyte of rabbit.

Therefore, in the broadest sense, the present invention involves thetransplantation of human or animal cells or cell nuclei into enucleatedoocytes of a species different from the nuclear donor to obtain nt-unitsat various pre-implantation stages for isolating ntES cells.

Human Cell Nuclear Transfer Technique

Nuclear transfer technique or nuclear transplantation techniques havebeen described in many references, such as some of the references citedin the background of the invention (See in particular, Wilmut, et al.,Nature 385:810-813, 1997; Campbell, et al., Biology of Reproduction 49(5): 933-942, 1993; Collas, et al., Mol. Reprod. Dev, 38:264-267, 1994;Keffer, et al., Biology of Reproduction, 50:935-939, 1994; Sims, et al.,PNAS, 90:6155-6159, 1993; and Patents NOs WO 94/26884, WO 94/24274, andWO 90/03432, which are incorporated by reference in their entiretyherein.

Nuclear Donor

In the invention, the cells used as donors for nuclear transfer arederived from human cells, preferably human fibroblasts.

Human or animal cells, preferably somatic cells, may be obtained andcultured according to the methods known in the art. Human and animalcells useful in the present invention include, by way of examples,epithelial cells, neural cells, epidermal cells, keratinocytes,hematopoletic cells, melanocytes, chondrocytes, lymphocytes (B and Tlymphocytes), nucleated erythrocytes, macrophages, mononuclear cells,fibroblasts, cardiac muscle cells, and other muscle cells. Moreover, thehuman cells used for nuclear transfer may be obtained from differentorgans, for instance, the skin, lung, pancreas, liver, stomach,intestine, heart, reproductive organs, bladder, kidney, and urethra andother urinary organs. Suitable donor cells (i.e., cells useful in thesubject invention) may be obtained from any cell or organ of the body,including all somatic cells.

Oocytes

The oocytes used for nuclear transfer may be obtained from variousanimals, including mammals and amphibians. Suitable mammalian sourcesfor oocytes include sheep, cattle, pigs, horses, rabbits, guinea pigs,mice, hamsters, rats, primates, etc. In a preferred embodiment, theoocytes will be obtained from a leporid source, most preferable from arabbit.

Mature metaphase II stage oocytes can be collected surgically from thereproductive tract of non-superovulated or superovulated rabbits 14 to24 hours after onset of estrus or the injection of human chorionicgonadotropin (hCG) or a similar hormone, with the optimal time being 15to 18 hours.

Methods for isolation of oocytes are well known in the art. Essentially,the method comprises isolating oocytes from the ovaries or reproductivetract of a mammal or amphibian, e.g. a rabbit.

The degree of maturation of the oocyte in nuclear transfer has beenreported to be a major factor in the success of the nuclear transfermethods (see, Prather et al., Differentiation, 48, 1-8, 1991). Ingeneral, previous successful mammalian animal cloning practices use themetaphase II stage oocytes as the recipient oocyte because at this stageit is believed that the oocyte can effectively “activate” the introducednucleus to initiate and develop to animal embryos.

Enucleating

It has been discovered in the present invention that mature oocytesobtained from New Zealand rabbits should be enucleated 15 to 23 hoursafter the injection of hCG. Prior to enucleating, the oocytes will beplaced in M2 culture medium (Sigma) containing hyaluronidase. Cumuluscells will be removed by repeated pipetting through pipettes very smallinner diameter or by vortexing briefly. The stripped oocytes are thenscreened for those contain polar bodies, and the selected metaphase IIoocytes, as confirmed by the presence of polar bodies, are then used fornuclear transfer, enucleating.

Generally, immature oocytes collected from animal ovaries should bematured in vitro as desired, until they are in the metaphase II stage.

For New Zealand rabbits, enucleating should be performed not more than20 hours past the injection of hCG, with 16 to 18 hours beingpreferable.

Enucleating may be accomplished micro-surgically using a micropipette toremove the polar body and the adjacent cytoplasm. The efficiency ofenucleating may be examined by staining the removed polar body andchromatin with Hoechst 33342 dye, and observed DNA under ultravioletirradiation rapidly.

nt-Unit Preparation

nt-units may be prepared according to the methods known per se in theart, e.g. by injection into the zona pellucid and by injection into thecytoplasm.

Injection into the Zona Pellucid:

A single animal or human cell or cell nucleus, which is typically of aspecies different from that of the enucleated oocyte, will betransferred into the perivitelline space of the latter. Preferably, thent-unit, consisting of a human or animal cell and a rabbit oocyte, willbe electrofused in a 0.5 mm chamber by 1-2 applications of an electricalpulse of 90-120V for about 60 μsec each or more frequently inelectrofusion medium (e.g. mannitol, sucrose or sorbitol fusion medium)16 to 20 hrs after the injection of hCG. After fusion, each fusednt-unit will be placed in a suitable tissue culture medium, e.g. RD{DEME (Gibco): RPMI-1640 (Gibco)}; M199 (Gibco), DMEM (Gibco), untilincubation.

Electrofusion is accomplished by providing a pulse of electricitysufficient to cause a transient breakdown of the plasma membrane.Essentially, if two adjacent membranes are induced to break down, thelipid bilayers will intermingle and small channels will open between thetwo cells after the membranes reform. Due to the thermodynamicinstability of such a small opening, it enlarges until the two cellsfuse into one, Reference is made to U.S. Pat. No. 4,997,384 by Pratheret al. (incorporated herein by reference in its entirety), whichprovides a further discussion of the process. A variety of electrofusionmedia can be used, including, e.g. sucrose, mannitol, sorbitol andphosphate buffered solution. Fusion can also be accomplished by usingSendai virus as a fusogenic agent.

According to the present invention, the nt-units may be activated byknown methods, including culturing the nt-units at sub-physiologicaltemperature, essentially by applying a cold temperature shock to thent-units. This may be most conveniently done by culturing the nt-unitsat room temperature, which is low relative to the physiologicaltemperature conditions to which embryos are normally exposed.

Suitable oocyte activation methods are the subject of U.S. Pat. No.5,496,720 by Susko-Parrish et al., which is herein incorporated byreference.

Oocyte activation may be effected sequentially:

-   (i) increasing levels of divalent cations in the oocyte, and-   (ii) reducing phosphorylation of cellular proteins in the oocyte.

The methods of increasing divalent cation levels include, for example,the addition of kinase inhibitors, e.g. the serine-threonine kinaseinhibitors 6-dimethyl-amino-purine, staurosporine, 2-aminopurine, andsphingosine.

Alternatively, phosphorylation of cellular proteins may be inhibited bythe introduction of a phosphatase into the oocyte (e.g. phosphatase 2Aor phosphatase 2B).

Injection into the Cytoplasm:

Other methods may be used for nuclear transplantation, includinginjecting the nucleus directly into the oocyte cytoplasm rather thanusing electroporation. These techniques are applicable only when thenucleus of the donor somatic cell are able to be reprogrammed by theoocyte, which in turn must be capable of inducing a human somatic cellreprogrammed in rabbit oocyte cytoplasm. (Collas and Barnes, Mol.Reprod. Dev, 38:264-267, 1994).

nt-Unit Culturing

An activated nt-unit may be cultured in a suitable culture medium forfurther development. For example, activated nt-units may be transferredinto and micro-cultured in medium RD, M199, DMEM50, 50 microliter ofculture medium being covered by a layer of paraffin, e.g. at 38° C. and5% CO₂. In one of the preferable examples, the highest rate ofblastocysts was obtained by culturing nt-units in RD medium.

According to the inventor's experience, for human cell/enucleated rabbitoocyte derived nt-units, the blastocyst will be obtained about 6-7 daysafter initiation of oocyte activation. nt-units will typically exhibitappearance and cellular characters similar to embryos of the nucleardonor species rather than the oocyte donor species. For example, in thecase of a nt-units obtained by the transfer of a human nuclear donorcell Into an enucleated rabbit oocyte, the development of the nt-unitfollows a schedule more typical of a human rather than a rabbit embryo.It takes approximately 6-7 days to form a blastocyst, unlike a rabbitembryo, which usually forms a blastocyst in approximately 3-4 days.

The media used for tissue culture and for maintaining rabbit embryosinclude DMEM+15% FBS; M199+15% FBS; and RD+15% FBS. In addition, theycan be used for co-culture with a variety of cell types, includinggranulose, oviduct, uterine, and STO cells.

ntES Cell Line Establishment:

The culture system is the most important factor in the ntES cell lineestablishment. The system includes the mediums and the feeder layers.The mediums mean a liquid suitable for ntES cells culturing, includingas a ingredient DMEM (Gibco); FBS (Hyclone); non-essential amino acidstock (Gibco); β-mercaptoethanol; Knockout medium (Gibco); SR (Gibco);and various factors.

The first type of the factors is the ligand of glucose-protein 130, e.g.LIF (R&D), which, together with the gp-130, may initiate the pathway ofsignal transduction.

The second type of the factors is the endogenous cAMP agonist, e.g.Forskoline (Sigma), preferably at 10 μM.

The third type of the factors is the growth factor, e.g. bFGF (R&D),which may inhibit the apoptosis of the ES cells.

The feeder layers mean the fibroblasts obtained from the 13.5 days'mouse embryo. They may sustain the ntES cells' growing afterinactivation by Mitomycin C (Sigma). In a preferred embodiment, thefeeder cells comprise mouse embryonic fibroblasts. The preparation of asuitable fibroblast feeder layer will be described in the examplesbelow.

The human ntES cells line was successfully obtained from the nt-unitsusing the above culture system. The human ntES cell colonies have alonger doubling time than the mouse ES cells, and exhibit colonyappearances and growth characters similar to human ES cells rather thanmouse ES cells.

The human ntES cells obtained according to the present invention sustainthe high positive for alkaline phosphatase (Sigma) after a long term ofculture, over 30 passages. They also express the surface markers incommon with the primate ES cells, e.g. SSEA-1 (−), SSEA-3 (+), SSEA-4(+), TRA-1-10 (+), and TRA-1-81 (+). This shows that the ES cellssustain growth in an undifferentiated state.

The human ntES cells obtained according to the present invention incolonies are centralized and compacted, and cells arranged closely, withbigger nucleus and less cytoplasm. They have the same growth charactersas the primate ES cells as follows:

(i) sustaining growth in an undifferentiated state in vitro,

(ii) maintaining normal karyotype during a long term of culturing, and

(iii) capable of developing into specific cell types of 3 germ layers,including ectoderm, mesoderm, and endoderm.

Induction and Differentiation:

Thomson, et al. (Science, 282 (6), November; 1145-1147, 1998) reportedthe successful establishment of the human ES cell line using theblastocysts remained from IVF. Then they also demonstrated that thehuman ES cells and the cells derived therefrom had the capability offorming the embryold body (ED) and derivatives of all three embryonicgerm layers, including ectoderm, mesoderm, and endoderm.

Wakayama, et al. (Science, 292 (5517): 740-743, 2001) reported thesuccessful isolation of the mouse ES cells from the blastocyst bysomatic cell nuclear transfer. The ES derived by somatic cell could beinduced to all kinds of specific cells in vitro. His experiment provedthe blastocyst from nuclear transfer has the same use of deriving the EScells as the normal blastocyst. The ntES derived by somatic cell had thefully pluripotency, capable of developing into an integral adult and allcell types contained therein.

The invention provides a method to produce the ntES cells by nucleartransfer. The studies conducted by the inventors demonstrate that thehuman ntES cells have the same capabilities, like the normal ES cellsderived; forming the embryold body and differentiate to all threeembryonic germ layers (e.g. ectoderm—neurofilament, NSE: mesoderm—myoD,myoglobin, desmin, vWF; endoderm—α-anti-trypsin, α-fetoprotein molecularmarkers positive).

The invention provides various conditions for inducing human ntES cellsto differentiate, including inducing both in vitro and in vivo. Theinduction in vitro can be separated into (A) self-induction, whichinvolves the induction of the ntES cells or ntES-like cellsautomatically under the specific culturing conditions, and (B) thebiochemical induction, which involves putting the ntES cells or ntEScells into the mediums comprising retinoic acid or beta-mercaptoethanol(Sigma) or DMSO or H₂O₂ for the first stage and then replacing themedium with other special medium to promote the cells differentiation.

Induction in vivo involves putting the human ntES cells directly orindirectly, after being induced in vitro, into the special parts ofanimal or human and inducing them to differentiate,

The Prospects of ntES Cell:

The human ntES cells, embryonic stem-like cells or other types ofembryo-derived stem cells obtained from the nt-units in the presentinvention could be used in the therapy of many diseases/many therapeuticusages.

In principle, the ntES cells may be used to obtain any desireddifferentiated cell type. Therapeutic usages of such differentiatedhuman cells are unparalleled. For example, human hematopoietic stemcells may be used in medical treatments requiring bone marrowtransplantation, and human neural stem cells may be used in regenerativemedicine for spinal cord injuries and Parkinson's disease.

Other diseases and conditions treatable by isogenic cell therapyinclude, by way of example, multiple scleroses, muscular dystrophy,diabetes, liver diseases, heart diseases, cartilage replacement, burns,foot ulcers, gastrointestinal diseases, vascular diseases, kidneydiseases, urinary tract diseases, and aging related diseases andconditions.

The human ntES cells or the derived cells thereof produced according tothe present invention may be used as cell carriers to transport allkinds of extrinsic bio-functional material into human body. Transfer theDNA, RNA protein or other bio-functional materials into the ntES cellsor its derived cells thereof by the methods of transgene, homologousgene recombination, transposon plasmid transfection, virus transfection,etc, and then transfer the ntES cells or the derived cells thereof intothe human body, thus the DNA, RNA and protein transferred can play arole in vivo.

By using the method as described above, the defective genes, e.g.defective immune system genes, cystic fibrosis genes can be replaced, orthe genes which can express therapeutically beneficial proteins such asgrowth factors, lymphokines, cytokines, enzyme, etc, can be introduced.For example, the gene encoding brain growth factors may be introducedinto human embryonic stem cell or the derived cells of other types andthen the differentiated or undifferentiated cells genetically modifiedmay be transplanted into a patent suffering from Parkinson's disease totreat the disease.

Using such methods, desired genes may be introduced into the subjectntES cells, and the cells will differentiate into needed cell types,e.g. hematopoietic cells, neural cells, pancreatic cells, cartilagecells, etc, and then the resultant cells or the cells derived therefromcan be used in the therapy.

Genes which may be introduced into the subject ntES cells include, byway of example, epidermal growth factor, basic fibroblast growth factor,glial derived neurotrophic growth factor, insulin-like growth factor (Iand II), neurotrophin-3, neurotropphin-4/5, ciliary neurotrophic factor,AFT-1, cytokine genes (interleukins, interferons, colony stimulatingfactors, tumor necrosis factors (alpha and beta), etc.), genes encodingenzymes etc.

The subject ntES cells also may be used as the controlling genes and forthe study of genes, which are involved in the regulation of earlydevelopment to find out the important factors in the process of celldifferentiation. Abnormity differentiation and division of cells causedmany serious diseases and congenital malformationare. Therefore,in-depth studies on the process of differentiation, division andinduction of the normal cells will provide a clear understanding of thecells pathological process of those diseases.

Also, differentiated cells tissues and organs from the subject ntEScells may be used in the development of drugs. The study on theembryonic stem cells will greatly change the methods of producing drugsand safety examination for drugs. The embryonic stem cells maydifferentiate varied cells to be used in studying, screening andidentification of the drugs. In future drug research, only theexperimental drugs, which have passed the ES cell experiment in vitro,could be used on the experimental animal or used in the clinicalexperiment on human.

In addition, nuclear xeno-transplantation techniques may be used to savethe species close to extinction, such as the giant panda. In thesespecies, larger numbers of female oocytes are hardly to obtain as thenumber of the female is less. Their somatic cell may therefore betransplanted into enucleated oocytes of different species. The resultantnuclear transfer units would then be cultured in vitro to obtainblastocysts, which could then be transplanted into the pregnant motherto develop a normal individual.

In order to more clearly describe the invention, the following examplesare provided.

EXAMPLE 1 The preparation of nuclear donor cells for nuclear transfer

Foreskin tissue obtained from surgery with informed consent was mincedand washed with PBS, centrifuged at 1000 rpm for 5 minutes, digested by0.05% Trypsin/0.02% EDTA (Gibco) at 37° C. for 30 minutes. Removeexcessive solution from the tube and centrifuge the tube at 1000 rpm for5 minutes. Discard the supernatant and culture the cell pellet in 90%DMEM (Gibco)+10% FBS (Hyclone)+50 IU/ml penicillin-streptomycin (Gibco).Re-suspend in plate and incubate at 37° C., 5% CO₂, with the mediumchanged every 3 days. Passage after the cell grows to confluent and thecells of the 7^(th)-20^(th) passage are used as nuclear donor cells(FIG. 1).

Oocyte Preparation

Mature New Zealand female Rabbit, 2.5-3 kg, was superovulated with asingle dose injection of 100 IU PMSG (i.m.) (The firstbio-pharmaceutical company, Shanghai), followed by a single doseinjection of 100 IU hCG (HuaFu High Bio-Technology Company, Tlanjing)(i.v.) 72 hours later. Mature oocytes were flushed out of the oviducts14-16 hours after hCG injection using pre-warmed M2 solution (Sigma)Oocytes were put into a solution containing 300 IU/ml hyaluronidase toremove cumulus cells. The oocytes (FIG. 2) were then washed in M2solution for 3 times.

Nuclear Transfer Procedures

Injection into the Zona Pellucid:

Oocytes were manipulated in M2 medium with 7.5 μg/ml Cytochalasin B(Sigma) and incubated and enucleated by a needle with a bevel aftermaintained for 10 minutes at room temperature. After that, put thesingle donor fibroblast into the perivitelline space of enucleatedrabbit oocyte, forming nt-units. The nt-units were equilibrated in afusion buffer solution containing 0.3 M Glucose (Sigma); 0.1 mM MgCl₂(Sigma); 0.05 mM CaCl₂ (Sigma) and stimulated with a single directcurrent pulse of HV 120V for 60 μsec. After stimulation, the nt-unitswere incubated in RD medium, consisting of DMEM 42.5% (Gibco), RPMI-164042.5% (Gibco), and 15% FBS (Hyclone). After 6-7 days, blastocysts wereobtained (FIGS. 3-6, 11).

Injection into the Cytoplasm

Oocytes were incubated in M2 solution with 7.5 μg/ml Cytochalasin B(Sigma) and enucleated by a needle with a bevel after maintained for 10minutes at room temperature. After that, put the single donor fibroblastinto the cytoplasm of the enucleated rabbit oocyte, forming a nt-unit.Many nt-units were cultured in RD medium, consisting of DMEM 42.5%(Gibco), RPMI-1640 42.5% (Gibco), 15% FBS (Hyclone). After 6-7 days,blastocysts were obtained (FIGS. 7-10).

EXAMPLE 2 Establishing Human Nuclear Transfer Embryonic Stem Cells Lines

nt-units at the blastocyst stage were obtained as described above andpipetted up and down gently using a glass needle (stretched from a glasspipe of 3 mm diameter) with a diameter smaller than the blastocyst tostrip zona pellucid. Then, the inner cell masses of the blastocysts wereisolated and plated onto feeder layers and cultured in 79% DMEM (Gibco),20% FBS (Hyclone), 1% non-essential amino acid stock (Gibco), 0.1 mMβ-mercaptoethanol (Gibco), 10 ng/ml LIF (R&D), 10 ng/ml bFGF (R&D), 10μM Forskolin (Sigma) at 37° C. in 5% CO₂, with half of the mediumchanged every 2 days.

After 2-4 days' culturing, cell mass was observed to be growing on thefeeder. After 7-20 days, colonies were observed (FIG. 12). The colonieswere dispersed by enzyme or mechanical and passed onto a platecontaining fresh feeder layer. After 20 passages, the ntES cells werecryopreserved.

Fibroblast Feeder Layer

The feeder cells were derived from mouse embryos of 14-16 days old.After the removal of the head, liver, heart and esophagus of the embryosunder sterile condition, the remains of the embryo were minced, digestedin a pre-warmed solution containing 0.05% Trypsin/0.02% EDTA (Gibco),incubate at 37° C. for 30 minutes, centrifuged at 1000 rpm for 5minutes. The cells pellet was re-suspended In 90% DMEM (Gibco); 10% FBS(Hyclone); 50 IU penicillin-streptomycin (Gibco), plated and incubatedat 37° C., 5% CO₂. After passage 3 times, a feeder layer was treatedwith 10 mM Mitomycin C (Sigma) for 3-4 hours and passed on 4-well or96-well plate. A feeder layer grew in a 37° C. humidified incubatorcontaining 5% CO₂. The plates with homogenous feeder layer were used toprepare S-ES Culture.

Charicterization of ntES Cells

Primate ES cells express alkaline phosphatase activity, and can expressa series of characteristic surface antigens, and therefore they can beidentified by detecting Alkaline phosphatase activity and theimmunohistochemistry of SSEA-1, SSEA-3, SSEA-4, TRA-1-10, TRA-1-81antibodies.

Method of detecting Alkaline phosphatase activity: The ES cells culturedon 4-well plate were fixed with 4% paraformaldehyde for 5 minutes, andwere washed with PBS for 3 times. Then, added the substrate of alkalinephosphatase (Sigma) to the well, and stay at dark for 15 minutes. ntEScells expressed a high level of alkaline phosphatase activity, but themouse feeder cells are negative. (FIG. 13).

Immunohistochemical method: Plated the ES cells onto a plastic glassslide, then fixed with 4% paraformaldehyde for 5 minutes, washed withPBS for 3 times, then added the 1:5 to 1:25 antibody dilutions ofSSEA-1, SSEA-3, SSEA-4, TRA-1-10, TRA-1-81 (available from DevelopmentalHybridoma Bank, Iowa City, Iowa) to the wells respectively, stayed atroom temperature for 1 hour, washed with PBS 2 times, add secondantibody-FITC, stay at RT for 30 minutes, washed with PBS for 2 times,added the second antibody labeled with FITC and cultured for 30 mins,and examined by a laser confocal microscope.

The result of 5 different antibodies detected: 4 are positive (FIG. 13).Antibody SSEA-1 SSEA-3 SSEA-4 TRA-1-10 TRA-1-18 Result − + + + +Karyotyping

Put 10 μg/ml Colchicine in cell cultures. After 37° C. for 4 hours, thecells were removed out from the plate and centrifuged cells at 1000 rpmfor 8 minutes. Discard the supernatant, re-suspend cell pellet inpre-warmed 0.05M KCl, and incubate at 37° C. for 30 minutes. Centrifugeat 1000 rpm for 8 mins, Discard the supernatant, re-suspend cell pelletin a fixation solution (methanol:ice acetic acid=3:1) and keep at roomtemperature for 15 minutes. Centrifuge at 1000 rpm for 8 minutes,Discard the supernatant, and re-suspend cells pellet again in a fixationsolution at room temperature for 15 minutes. After centrifuging at 1000rpm for 8 minutes, put cell pellet into a glass plate and stain withGIEMSA to examine the karyotype. One of he results is shown in FIG. 20.

EXAMPLE 3 Induction of Early Muscle Cells Differentiation

Culture the EB or EB-like cell mass in the RA medium containing DMEM(Gibco), 0.5 μm retinoic acid (Sigma), 10% FBS (Hyclone) for 5 days.After replacing the medium with the muscle cell culture mediumcontaining 79% DMEM (Gibco), 10% FBS (Hyclone), 10% Horse Serum (Sigma),1% chick embryo extract (Gibco), culture further 7-10 days, can becomemuscle-like cell. The cells arrange in parallel, some of which haveseveral nucleus (FIG. 15).

EXAMPLE 4 Inducution of Neuron Differentiation

Culture the EB or EB-like cell mass in the RA medium containing DMEM(Gibco), 0.5 μm retinoic acid (Sigma), 10% FBS (Hyclone) for 5 days.After replacing the medium with the neuron culture medium whichcontaining DMEM-F12 (Gibco), 1% ITS (Gibco), culture further for 7-10days, the EB or EB-like cell can become into neuron cells with somefilamentous projections stretching out from the cell body (FIG. 16).

EXAMPLE 5 Induction of Fibroblasts-Like Differentiation

Culture the EB or EB-like cell mass in the RA medium containing 90% DMEM(Gibco), 0.5 μm retinoic acid (Sigma), 10% FBS (Hyclone) or the neuronculture medium which containing DMEM-F12 (Gibco), 1% ITS (Gibco), or themuscle cell culture medium containing 79% DMEM (Gibco), 10% FBS(Hyclone), 10% Horse Serum (Sigma), 1% chick embryo extract (Gibco), theEB or EB-like cell can become into fibroblasts like cells. The cells areflat and polymorphous with big nucleus and dear nucleolus, and richcytoplasm (FIG. 17).

EXAMPLE 6 Induction of Adipocyte Differentiation

Culture the EB or EB-like cell mass in the RA medium containing 90% DMEM(Gibco), 0.5 μm retinoic acid (Sigma), 10% FBS (Hyclone) for severaldays, the EB or EB-like cell can become into adipocytes (FIG. 18). Thecells dyed by Oil Red O, an adipocyte-specific dye, are confirmed to beadipocytes containing fat drop in the cytoplasm.

EXAMPLE 7 Preparation of EB

Culture ntES cells in the ES cell medium containing 79% DMEM (Gibco),20% FBS (Hyclone), 1% non-essential amino acid stock (Gibco), 0.1 mMβ-mercaptoethanol (Gibco), 10 ng/ml LIF (R&D), 10 ng/ml bFGF (R&D), 10μM Forskolin (Sigma) at 37° C., 5% CO₂ for more than 7-14 days, aportion of ntES cells underwent spontaneous differentiation and formedEB (FIG. 14).

EXAMPLE 8 Identification of Three Germ Layers

Identify the EB or EB-like cell mass by an immunohistochemical method.

-   Ectoderm marker antibody: positive for nestin (Chemicon).-   Mesoderm marker: positive for myoglobin (Dako) and KDR (Sigma).-   Endoderm marker: positive for α-fetoprotein (BioGenex),    α-anti-trypsin (Dako).

The results suggested that the cell mass differentiated from EB orEB-like cell mass contained the cells derived from all three germ layerscells (FIG. 19).

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes and modification may be made withoutdeparting from the true spirit and scope of the invention. All suchmodifications are intended to be within the scope of the description andthe claims appended hereto.

1. A method of preparation of embryonic stem cells, embryonic stem-likecells or other types of embryo-derived stem cells by somatic cellnuclear transfer, comprising the following steps: I. transplantingmammalian cells or cell nuclei into enucleated oocytes to obtainnt-units; II. activating the obtained nt-units; III. culturing theactivated nt-units to obtain nt-units at various pre-implantationstages; and IV. isolating embryonic stem cells, embryo stem-like cells,or other types of embryo-derived stem cells from nt-units at adevelopmental stage a greater than the 2-cell stage.
 2. The method ofclaim 1, wherein said the donor cell is human somatic cell.
 3. Themethod of claim 1, wherein said the oocyte is from mammalian oramphibians.
 4. The method of claim 3, wherein said the oocyte is in themiddle of divisional period, preferable in metaphase II stage.
 5. Themethod of claim 3, wherein the said oocyte is enucleated within 24 hoursafter the injection of human chorionic gonadotropin.
 6. The method ofclaim 1, wherein the resultant nt-units are activated by culture at roomtemperature or by using an activating agent.
 7. The method of claim 6,wherein the activating agent is selected from a group consisting ofmannitol electrofusion solution, sucrose electrofusion solution,sorbitol electrofusion solution and phosphate buffered solution, morepreferably sucrose electrofusion solution.
 8. The method of claim 1,wherein the activated nuclear transfer units are cultured in a mediumselected from a group consisting of RD medium, M199 medium, and DMEMmedium, more preferably the RD medium, to obtain the nt-units at variouspre-implantation stages, including e.g. the 2˜4-cell, 8-cell, morula,blastocyst, and hatching blastocyst stages.
 9. The method of claim 1,wherein the activated nuclear transfer units are cultured in the mediumselected from a group consisting of RD medium, M199 medium, and DMEMmedium, and form the co-culture system with the multiple cells style,e.g. granular cell, oviduct cells, uterine cells and STO (mousefibroblast) cells, to obtain nt-units at various pre-implantationstages, including e.g. the 2˜4-cell, 8-cell, morula, blastocyst, andhatching blastocyst stages.
 10. The method of claim 1, wherein nt-unitsat the greater than 2-cell development stage are cultured to isolatentES cells, embryonic stem-like cells or other types of embryo-derivedstem cells under appropriate conditions.
 11. The method of claim 1,wherein the medium used to culture the ntES cells, embryonic stem-likecells or other types embryo-derived stem cells could be, but not limitedto, DMEM medium, or knockout medium, preferably DMEM medium.
 12. Themethod of claim 11, wherein the medium contains one or more otherfactors.
 13. The method of claim 12, wherein the other factor isselected from Leukemia Inhibitor Factor (LIF), basic fibroblast growthfactor (bFGF) and Forskolin.
 14. Nuclear transfer embryonic stem cells,embryonic stem-like cells or other types embryo-derived stem cellsobtained through somatic cell nuclear transfer according to anymethod(s) of claim 1
 15. A method of producing differentiated cellsderived from nuclear transfer embryonic stem cells or embryonicstem-like cells or other types embryo-derived stem cells, comprising thefollowing steps: I. obtaining nuclear transfer embryonic stem cells orembryonic stem-like cells or other types embryo-derived stem cells bysomatic cell nuclear transfer; and II. inducing the cells obtained instep I to differentiate to specific type of cells under appropriateconditions.
 16. The cell of claim 15, wherein the ntES cells, embryonicstem-like cells or other types embryo-derived stem cells can be inducedin the medium containing the reagent selected from a group consisting ofRA, DMSO, H₂O₂ and beta -mecaptoethanol.
 17. The method of claim 15,wherein embryonic stem cells or embryonic stem-like cells or other typesembryo-derived stem cells (including above cells without induction orafter spontaneous induction or biochemical induction) are cultured invarious media according to various specific cells.
 18. The method ofclaim 15, wherein all medium containing varied growth factorsrespectively or their combination induce embryonic stem cells orembryonic stem-like cells or other types embryo-derived stem cells. 19.The method of claim 15, wherein the ntES cells, embryonic stem-likecells or other types stem cells are transplanted into the animal andhuman's specific part and are being induced to differentiate intovarious cell types in the environment of the body.
 20. Nuclear transferembryonic stem cells, embryonic stem-like cells or other typesembryo-derived stem cells, obtained by the method of claim
 15. 21. Thedifferentiated cells of claim 20, which are human differentiated cells,of which the nuclear DNA is encoded by the transplanted human somaticcell.
 22. The differentiated cells of claim 21, which are derived frommore than 2-cell nt-units and its nuclear transfer embryonic stem cells,embryonic stem-like cells or other types of embryo-derived stem cellsthrough induction.
 23. The differentiated cells of claim 20, wherein anypart of the cells, e.g. nuclear DNA, mitochondrion, RNA, organelles,membrane etc. can be modified or changed artificially.
 24. The cells ofclaim 20, used as a carrier to bring artificially added molecules intothe body.
 25. The cells of claim 20, used for screening and evaluationof drugs.
 26. A treatment method that comprises administering to apatient differentiated human cells prepared according to claim
 20. 27.The method of claim 26, wherein said cell transplantation therapy iseffected for the treatment of a disease or condition selected from thegroup consisting of, for example but not limited to, Parkinson'sdisease, Huntington's disease, Alzheimer's disease, ALS, spinal corddefects or injuries, multiple sclerosis, muscular dystrophy, cysticfibrosis, liver disease, diabetes, heart disease, cartilage defects orinjuries, burns, foot ulcers, vascular disease, urinary tract disease,AIDS and cancer.